CHIP is a protein with cochaperone and E3 ubiquitin ligase activities that mediates the cellular stress response by directing misfolded proteins for refolding or degradation. CHIP-/- mice are intolerant to acute stress, demonstrate an advanced aging phenotype, and live half as long as normal mice. While CHIP is thought to be predominantly active in the cytosol, evidence suggests that CHIP noncanonically ubiquitinates nuclear substrates and may exhibit enhanced chromatin binding capability following acute stress. The goal of this proposal is to identify how CHIP participates in stress response within the nuclear compartment. The first aim of the proposal is to identify genomic regions that bind CHIP through chromatin immunoprecipitation with next- generation sequencing (ChIP-seq). Bioinformatics tools to identify common transcription factors and consensus motifs will be used to model CHIP-DNA interactions following stress. The promoters identified in this unbiased screen will be compared with preexisting mRNA microarray analysis to calculate relationships between promoter occupancy and gene expression and develop a comprehensive model of how CHIP regulates gene expression. The second aim of this proposal is to identify the functional consequences of the novel interaction between CHIP and Sirtuin 6 (SirT6), a telomere-associated histone deacetylase (HDAC) that targets H3K9 and thereby inhibits NF-:B-dependent and HIF11-dependent signaling. Preliminary evidence suggests that the CHIP-SirT6 interaction occurs under both basal and stressed conditions, and may serve to stabilize SirT6 protein, since SirT6 protein levels are reduced in cells and tissues lacking CHIP. To determine how CHIP affects SirT6 localization and subsequent HDAC activity, levels of acetylated H3K9 will be measured in normal and CHIP-depleted cells. An in vitro assay will be employed to determine whether CHIP-dependent ubiquitination of SirT6 affects SirT6 HDAC activity. Finally, ChIP-qPCR will be used to measure enrichment of SirT6 and acetylated H3K9 at gene promoters in normal and CHIP-depleted cells and tissues. In summary, using a combination of molecular biology, cell biology, and bioinformatics approaches, the goal of this proposal is to understand the role of CHIP in nuclear stress response by studying CHIP's potential to regulate transcription and the effect of CHIP's interaction with the sirtuin family member SirT6. PUBLIC HEALTH RELEVANCE: Cellular stress can damage proteins, and CHIP is a protein that can reverse stress by aiding in the refolding or degradation of misfolded proteins. The goal of this proposal is to determine whether CHIP is involved in stress- dependent regulation of gene expression and anti-aging signaling pathways. The regulation of stress response connects the biology of aging and the effects of acute exposure to toxic stimuli, and contributes significantly to a general understanding of lifespan extension and improving physiology with advancing age.